Supplementary Figure 4: Effects of SHP2 inhibition in KRAS-driven cancer cells.
(a) Comparison of the effect of SHP2 inhibition and KRAS knockdown on the proliferation of KRAS-mutant cancer cell lines bearing different oncogenic mutations in KRAS as indicated by color coding. The negative logarithm of RMC-4550 GI50 values for cancer cells bearing activating KRAS mutations (described in Supplementary Table 5) were plotted against the negative value of their Project Drive Sensitivity Score for KRAS shRNA knockdown. Cells with high sensitivity to KRAS knockdown have larger negative Sensitivity Scores, and cells with greater sensitivity to SHP2 inhibition have larger negative log(RMC-4550 GI50) values. (b-c) Effect of RMC-4550 on RAS-GTP and pERK levels in Hras−/−, Nras−/−, Kras−/− mouse embryonic fibroblasts (MEFs) expressing solely the indicated RAS or BRAF transcript. Cells were grown in 2D culture and incubated with 1 µM RMC-4550 for 24 h. b) Cellular lysates were prepared and levels of RAS-GTP and pERK determined by immunoblot. RAS-GTP was affinity purified from the cellular lysates using GST-tagged RAF-RBD coupled to glutathione agarose beads. (c) RAS-GTP levels were quantified in whole cell lysates via RAS-GTP ELISA. Significant reduction in RAS-GTP and pERK levels is observed for KRAS WT (WT-4B) and KRAS G12 expressing MEFs, while cells expressing KRASQ61H lack response. Values above bars represent fraction of RAS-GTP in RMC-4550 vs. DMSO treated cells. Data in (b) shows a representative example of three independent experiments; data in (c) shows a representative example of two independent experiments, and bars indicates mean of two technical replicates. (d-e) (d) Expression of the wild type SOS1 and SOS-F proteins was induced by doxycycline treatment for 24 h. Cells were treated with 50 ng/mL EGF at various time intervals, lysed, and total pERK levels were measured by AlphaLisa. The SOS-F construct showed increased and sustained levels of pERK signal in the absence of EGF stimulation. Shown is one representative example performed in technical duplicate of three independent experiments. Figure indicates mean +/− S.D. (e) NCI-H358 cells transiently expressing SOS1 and SOS-F encoding cDNA for 48 h were treated with vehicle or 1 µM RMC-4550 for 1 and 6 h. Harvested lysates from the cells were probed for the indicated proteins using western blot. Data shown represent at least 3 independent biological replicates. Source data is provided in Supplementary Table 9.
